Browsing by Subject "Chromatography, High Pressure Liquid"
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- ItemOpen AccessApparent Absorption Efficiencies of Nectar Sugars in the Cape Sugarbird, with a Comparison of Methods(1998) Jackson, Susan; Nicolson, Susan W; van Wyk, Ben‐ErikNectarivore sugar preferences and nectar composition in the Cape Floristic Kingdom (southern Africa) differ from trends reported for analogous systems in America and Europe in that sugarbirds and sunbirds show no aversion to sucrose, which is the dominant nectar sugar in many of their food plants. To elucidate the physiological bases (if any) of nectarivore sugar preferences, we determined apparent sugar absorption efficiencies in a passerine endemic to this region, the Cape sugarbird Promerops cafer. Apparent absorption efficiencies for the three major nectar sugars, sucrose, glucose, and fructose, were extremely high (> 99%), as in other specialized avian nectarivores. Xylose, a pentose sugar recently reported in the nectar of some Proteaceae, was absorbed and/or metabolized inefficiently, with a mean of 47.1% of ingested sugar recovered in cloacal fluid. We did not measure the proportions of xylose that were absorbed and/or metabolized. We also compared three methods of estimating absorption efficiency: (1) measurements of total sugar in cloacal fluid with refractometry, without correction for differences between volumes of ingesta and excreta; (2) the same measurements combined with correction for volume differences; and (3) HPLC analyses quantifying individual sugars in cloacal fluid, with correction for volume differences. Refractometry has been frequently used in previous studies. For all sugars except xylose, method 1 yielded results similar to those obtained with method 2, but the convergence was artifactual, and we do not recommend use of this method. Apparent absorption efficiencies calculated with method 2 underestimated true absorption efficiency, because refractometry measures nonsugar solutes, but this error is biologically significant only when efficiencies are low.
- ItemOpen AccessDNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts(1986) PARKER, M Iqbal; de Haa, Judy B; Gevers, WielandSodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.
- ItemOpen AccessIdentification of N -Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form(1997) Yu, X Christopher; Sturrock, Edward D; Wu, Zhuchun; Biemann, Klaus; EHLERS, Mario R W; Riordan, James FThe sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.